Receptor-triggered cytosolic calcium (Ca2+) signals are ubiquitously used in signal transduction. In most cells, an initial Ca2+ signals can be induced by receptor-mediated activation of phospholipase C, the production of the second messenger IPS, and the release of Ca2+ from ER Ca2+-stores. Nevertheless, this transient IPS-mediated Ca2+-signal is typically not sufficient to induce long-term regulation of transcription, secretion and other important cellular processes. In addition, cell activation requires a Ca2+-store depletion triggered opening of plasma membrane Ca2+ channels. Our grant investigates this important but poorly understood "store-operated Ca2+ signaling pathway" (SOC). Previous experimental evidence suggested that this ubiquitous signaling pathway begins in the lumen of the ER where an unkown Ca2+-sensor would monitor the loss of ER Ca2+ and then would signal to the plasma membrane to open more Ca2+-channels. During the last funding period, we discovered that STIM1 and STIM2 proteins function as Ca2+-sensors in the ER and that they are necessary and sufficient for signaling to the plasma membrane and opening SOC Ca2+-influx channels. This is the first molecular component that has been identified in this important cell signaling pathway. Our grant is focusing on these STIM proteins and on other regulators of the SOC signaling pathway that we have identified in subsequent siRNA screens using a human cell line. We will be using calcium imaging, fluorescence microscopy of protein distribution, biochemical approaches and mutant constructs of STIM and other identified regulators to dissect the mechanism of action of STIM proteins and other identified regulators in the regulation of SOC. We will also investigate the broader question of how this SOC signaling pathway participates in the balance of plasma membrane Ca2+-influx and Ca2+-export. Finally, we will develop a quantitative model of the global Ca2+ signaling network that integrates this new signaling pathway with other Ca2+-regulatory mechanisms. We will test our model by using siRNA perturbations and will compare the model predictions against measurements of the resulting changes in Ca2+-f luxes. Since this SOC influx pathway has been shown to be essential for many cellular processes such as T- and B-cell activation, mast cell activation and osteoclast differentiation, it is likely that our findings of new signaling components and regulatory processes in the SOC pathway will offer opportunities for developing novel drugs that improve pathological conditions such as transplant rejection, inflammation, allergies and autoimmune diseases.